Project Summary Many vaccine production and delivery systems remain dependent on a cold chain requirement, which prevents millions of people from receiving vaccines annually. To increase the availability of current and future vaccines, the vaccine cold chain needs to be eliminated. This project will use a bio-inspired approach to enhance molecular crowding for viral vaccines so they can remain stable in temperatures as high as 45C, which would support delivery in Africa. Molecular crowding will be induced by the use of polyelectrolytes that form dense coacervate phases. Peptides will be used as the polyelectrolytes because they are simple to synthesize and will break down easily in the body, providing a biocompatible storage system for live viral vaccines. An additional advantage to this system, is that it not only stabilizes viral vaccines, but it also concentrates the vaccine, thus eliminating one step in the manufacturing of large volumes of vaccine product. To understand and optimize the effect of polyelectrolyte chemistry on virus thermal stability, well-defined design rules related to the charge density, chemistry, and sequence of self-assembling peptides to maximize virus encapsulation and thermal stability will be determined. The disaggregation of the coacervates with biologically friendly buffers will also be a design criteria. Two hypotheses will be tested: 1) thermal stability of non-enveloped viruses can be enhanced by controlling the charge density of polyelectrolytes used to form dense phases and 2) enveloped viruses will require molecules to act as plasticizers to allow fluidity around the lipid membrane while confined in the dense polyelectrolyte phase. Osmolytes will be used as plasticizers in low concentration and their effect on dense phase formation will be determined. The hypotheses will be tested in two Aims. In Aim 1, design rules for encapsulating and stabilizing viruses using polyelectrolyte systems will be determined. Peptide sequence, charge density, and hydrophobicity will control encapsulation, virus stability, and disaggregation conditions. Aim 2 will determine the thermal stability, degradation pathways, and immune response of encapsulated virus particles. Traditional characterization, along with a novel method of chemical force microscopy (CFM) will be used to determine changes in virus infectivity, size, antibody adhesion and chemical surface changes after exposure to encapsulation. CFM will also be used to determine the interaction of the peptide polyelectrolytes with the virus, with and without osmolyte plasticizers. This will provide additional information on the function of osmolytes in current vaccine formulations. Four viruses will be studied, two enveloped and two non-enveloped, and both RNA and DNA viruses, to begin to establish design rules for a variety of viral vaccines. The immune response in vivo will be studied for one virus to confirm that coacervation does not change the response to the viral vaccine. The outcome of this project will provide a set of design rules for polyelectrolyte systems that thermally stabilize vaccines and can be released in biologically friendly buffers.